Fig 2: MEN1 knockdown promoted the expression of genes that regulate milk protein synthesis in mammary epithelial cells. The expression levels of factors involved in the PI3K/Akt/mTOR pathway (Akt, mTOR, S6K1 and 4E-BP1) that are associated with milk protein synthesis were assessed at the mRNA ((A) n = 3) and total protein ((B) n = 3) levels by quantitative real-time PCR (qRT-PCR) and WB, respectively, in MAC-T cells at 24 h after transfection with a non-specific control (control) or a MEN1 specific siRNA pool (siRNA). The phosphorylation levels of Akt at Ser473, mTOR at Ser2448, S6K1 at Thr389 and 4E-BP1 at Thr37/46 were detected simultaneously. The data are shown as the relative expression levels normalized to the internal control, β-actin. *P < 0.05, **P < 0.01. The horizontal dashed line represents the normalized level of their corresponding controls. Representative WB images of the expression of Menin, Akt, Akt phosphorylated at Ser473, mTOR, mTOR phosphorylated at Ser2448, S6K1 and S6K1 phosphorylated at Thr389 are shown in (C). PI3K, phosphatidylinositol-3-kinase; Akt, also known as protein kinase (B) Akt (Ser473), Akt phosplorylated at Ser473; mTOR, mammalian target of rapamycin; mTOR (Ser2448), mTOR phosplorylated at Ser2448; S6K1, p70 ribosomal protein S6 kinase-1; S6K1 (Thr389), S6K1 phosplorylated at Thr389; 4E-BP1, eukaryotic initiation factor 4E (eIF4E)-binding protein 1.

Fig 3: Effect of rapamycin on markers of the UPR pathways(A) Cells were incubated in medium alone (0.05% FBS), with 3 nM IGF-1 or with both 3 nM IGF-1 and 10 nM rapamycin (IGF-1 + rapamycin). Total protein extracts prepared from cells incubated for 24 h in those conditions were subjected to Western Blot analysis. Protein expression levels were assessed for phosphorylated or total forms of PERK, eIF2α and for CHOP, BiP, XBP1-s and XBP1-u proteins. Efficiency of rapamycin to inhibit mTORC1 pathway was also checked by immunoblot with phosphorylated and total forms of p70S6K1 and 4E-BP1. α-tubulin was used as internal control. (B and C) Cells were incubated with 10nM rapamycin for 1 h (B) or 24 h (C). Immunoblots for phosphorylated and total forms of PERK, eIF2α, p70S6K1 and 4E-BP1 or for ATF4, CHOP, BiP, XBP1-s and XBP1-u protein expression were performed. α-tubulin was used as internal control. Blots of P-p70S6K1, p70S6K1, P-PERK, PERK, and α-tubulin of Figure 4B and blots of Figure 4C have been performed on the same electrophoresis gel, but cut and reconstituted. (D) Cells were incubated in medium alone (Ctrl) or with 10 nM rapamycin for 24 h. Nuclear localization of ATF6 was assessed using immunofluorescence with ATF6-antibody (red) and Hoechst dye. Magnification ×1000. (E) Bar graphs were obtained by quantification of ATF6 nuclear staining. Results are representative of at least 3 experiments.

Fig 4: Upregulation of the mTORC1 signaling pathway in TSC2-/- hearts.The data at 4 months of age in (A) to (C). The data at 4 weeks of age in (D) to (F). (A) and (D): Western blot analysis of signaling proteins upstream or downstream of mTORC1 in the heart of TSC2+/+ or TSC2-/- mice. p-Akt, t-Akt, p-AMPK, t-AMPK, p-S6 and t-S6 indicate phosphorylated Akt, total Akt, phosphorylated AMPK, total AMPK, phosphorylated S6 and total S6, respectively. Data of phosphorylated proteins were normalized to corresponding total protein content, TSC1 and t-AMPK to alpha-tubulin and gamma-form to total 4E-BP1 (t-4E-BP1), respectively. (B) and (E): Western blot analyses of KDEL and ubiquitinated proteins. (C) and (F): Western blot analyses of LC3 and p62. Data were normalized to the alpha-tubulin protein. All data are expressed as fold increase over levels in the TSC2+/+ group. Open and closed bars represent TSC2+/+ and TSC2-/- mice, respectively. Values represent the mean ± S.E.M. of data from 3–7 mice in each group. *P < 0.05 versus corresponding control.

Fig 5: The endocrine hormones prolactin (PRL) and insulin (INS) modulated the expression of MEN1/Menin and its downstream factors involved in mTOR signaling. The MEN1 mRNA (A) and Menin protein (B) level were significantly inhibited in MAC-T cells after 8 h of treatment with prolactin and insulin, compared to their saline-treated controls. The factors in the mTOR pathway, Akt, mTOR, S6K1 and 4E-BP1, along with CSNK, were found to be upregulated upon prolactin (C) and insulin (D) treatment. As shown above, the data are relative expression levels normalized to the internal control β-actin. *P < 0.05, **P < 0.01. Akt, also known as protein kinase (B) mTOR, mammalian target of rapamycin; S6K1, p70 ribosomal protein S6 kinase-1; 4E-BP1, eukaryotic initiation factor 4E (eIF4E)-binding protein 1; CSNK, κ-casein.